Optimized Screening Methods for Investigation of the Larval Settlement of Lanice conchilega on Artificial Substrates
Round 1
Reviewer 1 Report
This manuscript examine various types of artificial substrate on the physical surface velocity and settlement of the polychaetes larvae. The design of the experiment is good and there are some issue need minor revision:
1) Introduction - I think the author should introduce more on the basic information and ecology of the polychaete worm Lanice. What are their habitats, what they feed, what their burrows look like and why they are eco engineerer. Even the introduction has not mention the target species is a polychaete worm.
2) Larval experiment - line 354 - what is mean by distribution of larvae. Why not count the number of juveniles or settlers in each replicate. I cannot understand what is the meaning of the distribution, very confusion. Figure 10, why express percentage of larvae in each treatment? Why not express the number of larvae settled and metamorphosed in each treatment? This figure need extensive revision.
3) This study is only based in lab, the discussion should elaborate how to establish such preferred substrate in the natural sea bottom. Will they be scour by sands? Totally cover by sand in a few days? Can they withstand strong wave in the Belgium sandy coast? This needed to be elaborate whether these substratum can really be used in the real life.
Author Response
This manuscript examine various types of artificial substrate on the physical surface velocity and settlement of the polychaetes larvae. The design of the experiment is good and there are some issue need minor revision:
1) Introduction - I think the author should introduce more on the basic information and ecology of the polychaete worm Lanice. What are their habitats, what they feed, what their burrows look like and why they are eco engineerer. Even the introduction has not mention the target species is a polychaete worm. ADH: General information and additional references were added between lines 60 to 65 in the revised manual.
2) Larval experiment - line 354 - what is mean by distribution of larvae. Why not count the number of juveniles or settlers in each replicate. I cannot understand what is the meaning of the distribution, very confusion. Figure 10, why express percentage of larvae in each treatment? Why not express the number of larvae settled and metamorphosed in each treatment? This figure need extensive revision.
ADH: Different numbers of individuals were added to the tanks at the beginning of the first (110 individuals) and second (85 individuals) trials. Therefore, we could not compare the result in number of individuals. We chose to express the distribution of larvae in each substrate (or the occupancy rate of each substrate) as a percentage of surviving individuals at the end of the experiment. I added this information in the figure caption as well as in the material and method for more clarity. Material and method: L339-343 and figure L431
3) This study is only based in lab, the discussion should elaborate how to establish such preferred substrate in the natural sea bottom. Will they be scour by sands? Totally cover by sand in a few days? Can they withstand strong wave in the Belgium sandy coast? This needed to be elaborate whether these substratum can really be used in the real life.
ADH: Indeed, this is an important issue. Some preliminary field testing had been done with the geotextiles used in this study. I added a paragraph referring to this study in the discussion, in the conclusion and future prospect section: “Previous field trials elaborate more on the specific techniques used in placing the substrates in the field at the low waterline, by a specific design to maintain the substrates as close as possible near the sediment[2], [30]–[32], [34]. Preliminary trials using the same 3D geotextiles used in this study gave interesting results and opened the possibility of effectively applying this technique in the field [34]. “ This paragraph can be found L.664-669 of the revised manuscript.
ADH: Overall, I wanted to thank you for taking the necessary time and effort to review the manuscript. I sincerely appreciated your comments and suggestions which, I believe, helped improve the quality of the manuscript.
Reviewer 2 Report
The study evaluates artificial screening methods for optimised larval settlement of the burrowing marine polychaete Lanice conchilega. The study is rooted in the context of nature-based coastal protection against erosion along the sandy shores of Belgium. Laboratory-based evaluation of larval settlement enhancing substrates is presented as an alternative to traditional in situ evaluation methods. The motivation was that the laboratory environment allows for not only controlled experimental conditions, but also for more substrates to be evaluated simultaneously. Another innovative element involves the combined use of both live larvae and passively floating plastic particles, which in my opinion, would be difficult to replicate in the field. Artificial substrates were assessed on how water movement (defined here as “currents”) and larval settlement were influenced, depending on substrate type and structure. The study reports significant effects related to both currents and particle/larval settlements and concludes with a number of recommendations of optimal substrate configurations and considerations for future examinations of this kind.
The study was generally well framed, methods clearly communicated and results adequately illustrated. I particularly appreciated the discussion on limitations of the methods employed, which gives context to the conclusions made. Overall, I would suggest the authors are making a notable contribution with implications for future work. However, the following points needs to be addressed before the article is accepted for publication.
The article will benefit from additional attention to grammar and editorial aspects (e.g. italisation of species names, see Line 55), standardised referencing of units (e.g. Line 57 “m-2”) etc. Also, the correct use of some terms should be reviewed. For example, consider replacing “replica” and “replicas” with “replicate” and “replicates”.
Specific comments:
Abstract:
Lines (L) 21-23: I suggest the quotation marks be eliminated and the sentence rephrased as follows: “Of the eight substrates evaluated, one proofed to significantly enhance the settlement of L. conchilega, namely: Geotextile 3D knitted fabric with PES knit, PA spacers and wood sticks mounted at a density of 680 sticks/m2”.
Keywords:
L26-27: I suggest avoiding duplicating words/phrases already listed in your title. For example, both "Lanice conchilega" and "larval settlement" can be eliminated or replaced.
Introduction:
L55: shuffle words to read: “the use of artificially enhanced biogenic reefs”.
L64-66: join sentences to read: “...which undergo a short benthic transition into the pelagic phase as aulophore larvae”
L70-71: Instead of “have tackled”, consider “Selected studies have addressed this question through in situ assessments of different types of epibenthic holdfast structures, which included...”
L84-85: This is the first mention of two key variables, namely: currents and larval settlement. The reader would benefit from a brief interlude into the significance and relevance of these variables. One could introduce the effect of currents on larval settlement as well as how currents are influenced by substrate structure. Yes, there is an elaboration in the Discussion section, but until then not much context is given.
L85-86: Your expansive efforts to compare multiple substrates in the laboratory setting forms a key part of the significance of your contribution. According to L85-86, past attempts to study aspects of L. conchilega larvae in the laboratory has been just that, attempts? An elaboration on this would strengthen your case, or at the least give context to the success of your experimental design, notwithstanding the shortcomings regarding mortality rates mentioned elsewhere.
L88-89: Did you qualify the accuracy of your substrate comparisons? I suggest eliminating this word entirely from the sentence to instead read simply: “...conditions to compare a large range of substrates..” or even better, instead of “large range”, consider the exact number of substrates.
Materials and Methods
The order of figures ought to be reviewed. L98 mentions the first reference to a figure, which in the present configuration reads Fig. 3, followed by Fig. 1a in L109 and so on. I suggest ordering your figures according to mentions in the body of text.
L106: substitute “tackled” with “addressed”
L123: change “to standardise” to “standardisation of”
L128-129: perhaps consider the following in the balance of comments from other reviewers and guidance from the editorial team, but as far as I’m concerned, the standardised expression of salinity is now unitless, PSU is not widely applied in this sense anymore. It should read “The salinity was 34±1” and please qualify the error in this sense. What are you reporting ±1SD, ±2SD or SE? I’m assuming you are referring to average values, so therefore “The mean salinity (±1SD) was...”
L150: Fig. 2 caption change “substrate” to “substrates”
L174: I’m assuming the volume of 25 mL relates to pellets and not the tank, so therefore “...volume of pellets (25 mL) was added to the tank in front..”
L183: Figure 4 axes, notation and key fonds to be enlarged.
L185: Numeral at the start of a sentence should written out rather, i.e. “Four substrate types”
L204: The significance of the specific frequency of readings made, i.e. 0.066 s is not adequately explained. Please elaborate.
L216: “recommendation” should be plural
L222: Why the variation in the number of replicates? Please elaborate.
L255: name the vessel appropriately, i.e. Research Vessel Simon Stevin or RV Simon Stevin
L291: Change sentence to “In the instance of a significant result, the one-way ANOVA was followed by the Tuckey HSD test to allow for subsequent pairwise comparison tests.
Discussion
L369: change “set-up” to “design”
L425: should be written in past tense “the aim was” and rephrased to better emphasise the point: “Finally the aim was to test the retention efficiency of substrates with living specimens, as a necessary step before recommending one particular substrate for in situ deployment”
L426-428: As with the preceding sentence, I suggest “Therefore, we opted to perform these tests with L. conchilega as a means to validate the hypothesis emerging from results obtained in the previous ranking tests”. At this point, it may be necessary to confirm exactly what this hypothesis is you are mentioning.
L438: The mention of the presence of Phaeocystis needs context. If you are referring to DMS production, then please state so explicitly with a relevant reference. I suggest as a starting point: Liss, P.S., Malin, G., Turner, S.M., Holligan, P.M., 1994. Dimethyl sulphide and Phaeocystis: A review. Journal of Marine Systems 5, 41–53. https://doi.org/10.1016/0924-7963(94)90015-9
L519-522: Is this not contradicting earlier statements? In L499-507 a case is made for how efficiently structure, like the application of sticks, are reducing flow, which in turn enhances attachment and settlement rate, while in L519-522 the authors link the superior performs of substrate G with the combined effects of increase flow and geotextiles. The premise in the latter hypothesis suggest higher flow should increase the number of larvae moving through the structure, which then equates to higher settlement and increased success of the substrate. Even so, it still contradicts the earlier statement. Which is best, low or higher flow? Surely the structure induced by the addition of sticks and the like would in fact reduce the flow of currents, thereby enhancing the ability of larvae to settle (further enhanced by the presence of vertical structures, mimicking their natural settling behaviour in the presence of tubes).
Author Response
The study evaluates artificial screening methods for optimised larval settlement of the burrowing marine polychaete Lanice conchilega. The study is rooted in the context of nature-based coastal protection against erosion along the sandy shores of Belgium. Laboratory-based evaluation of larval settlement enhancing substrates is presented as an alternative to traditional in situ evaluation methods. The motivation was that the laboratory environment allows for not only controlled experimental conditions, but also for more substrates to be evaluated simultaneously. Another innovative element involves the combined use of both live larvae and passively floating plastic particles, which in my opinion, would be difficult to replicate in the field. Artificial substrates were assessed on how water movement (defined here as “currents”) and larval settlement were influenced, depending on substrate type and structure. The study reports significant effects related to both currents and particle/larval settlements and concludes with a number of recommendations of optimal substrate configurations and considerations for future examinations of this kind.
The study was generally well framed, methods clearly communicated and results adequately illustrated. I particularly appreciated the discussion on limitations of the methods employed, which gives context to the conclusions made. Overall, I would suggest the authors are making a notable contribution with implications for future work. However, the following points needs to be addressed before the article is accepted for publication.
ADH: Overall, I wanted to thank you for taking the necessary time and effort to deliver an in-depth review of the manuscript. I sincerely appreciate your very clear comments and suggestions which, I believe, helped in improving the quality of the manuscript. I took them all into consideration with pleasure.
The article will benefit from additional attention to grammar and editorial aspects (e.g. italisation of species names, see Line 55), standardised referencing of units (e.g. Line 57 “m-2”) etc. Also, the correct use of some terms should be reviewed. For example, consider replacing “replica” and “replicas” with “replicate” and “replicates”. ADH: The changes were made.
Specific comments:
Abstract:
Lines (L) 21-23: I suggest the quotation marks be eliminated and the sentence rephrased as follows: “Of the eight substrates evaluated, one proofed to significantly enhance the settlement of L. conchilega, namely: Geotextile 3D knitted fabric with PES knit, PA spacers and wood sticks mounted at a density of 680 sticks/m2”. ADH: The change was made.
Keywords:
L26-27: I suggest avoiding duplicating words/phrases already listed in your title. For example, both "Lanice conchilega" and "larval settlement" can be eliminated or replaced. ADH: "Lanice conchilega" was replaced by "ecosystem engineer" and "Larval settlement" by "Aulophora".
Introduction:
L55: shuffle words to read: “the use of artificially enhanced biogenic reefs”. ADH: The change was made.
L64-66: join sentences to read: “...which undergo a short benthic transition into the pelagic phase as aulophore larvae” ADH: The change was made.
L70-71: Instead of “have tackled”, consider “Selected studies have addressed this question through in situ assessments of different types of epibenthic holdfast structures, which included...” ADH: The change was made.
L84-85: This is the first mention of two key variables, namely: currents and larval settlement. The reader would benefit from a brief interlude into the significance and relevance of these variables. One could introduce the effect of currents on larval settlement as well as how currents are influenced by substrate structure. Yes, there is an elaboration in the Discussion section, but until then not much context is given. ADH : I Added “Characterising the influence of the substrate on both hydrodynamics and capture rate is important because velocity can influence settlement [39] and the capture capacity is an essential tool for colonization of defaunated substrates [40]. “ L 99-102 of the revised manuscript.
L85-86: Your expansive efforts to compare multiple substrates in the laboratory setting forms a key part of the significance of your contribution. According to L85-86, past attempts to study aspects of L. conchilega larvae in the laboratory has been just that, attempts? An elaboration on this would strengthen your case, or at the least give context to the success of your experimental design, notwithstanding the shortcomings regarding mortality rates mentioned elsewhere. ADH: The sentence “An attempt of developing laboratory experiments using L.conchilega larvae [33] has been done, emphasising the difficulties to screen substrates using live organism.” was changed to read “A previous study [33] has made an attempt to develop laboratory experiments for studying settlement of L. conchilega larvae on artificial substrated, but no significant preference was found between the tested substrates, emphasising the difficulties to screen substrates using live organisms in laboratory context.” L102-105 of the revised manuscript.
L88-89: Did you qualify the accuracy of your substrate comparisons? I suggest eliminating this word entirely from the sentence to instead read simply: “...conditions to compare a large range of substrates..” or even better, instead of “large range”, consider the exact number of substrates. ADH: The word accuracy was removed, and the number of substrates was added.
Materials and Methods
The order of figures ought to be reviewed. L98 mentions the first reference to a figure, which in the present configuration reads Fig. 3, followed by Fig. 1a in L109 and so on. I suggest ordering your figures according to mentions in the body of text. ADH: The changes were made.
L106: substitute “tackled” with “addressed” ADH: The change was made.
L123: change “to standardise” to “standardisation of” ADH: The change was made.
L128-129: perhaps consider the following in the balance of comments from other reviewers and guidance from the editorial team, but as far as I’m concerned, the standardised expression of salinity is now unitless, PSU is not widely applied in this sense anymore. It should read “The salinity was 34±1” and please qualify the error in this sense. What are you reporting ±1SD, ±2SD or SE? I’m assuming you are referring to average values, so therefore “The mean salinity (±1SD) was...” ADH: The sentence “ The salinity was 34±1 PSU (salinity refractometer” was changed to read “The mean salinity (±1SD) was 34 (salinity refractometer).”
L150: Fig. 2 caption change “substrate” to “substrates” ADH: The change was made.
L174: I’m assuming the volume of 25 mL relates to pellets and not the tank, so therefore “...volume of pellets (25 mL) was added to the tank in front..” ADH: The change was made.
L183: Figure 4 axes, notation and key fonds to be enlarged. ADH: The change was made.
L185: Numeral at the start of a sentence should written out rather, i.e. “Four substrate types” ADH: The changes were made for all figures.
L204: The significance of the specific frequency of readings made, i.e. 0.066 s is not adequately explained. Please elaborate. ADH: The frequency of reading was chosen based on the Nortek supplier recommendations. I added the reference to the supplier manual in the manuscript. The choice of the frequency was made based on the obtention of the best data quality while remaining compatible with the processing capacity of the computer used in the experiment.
L216: “recommendation” should be plural ADH: The change was made.
L222: Why the variation in the number of replicates? Please elaborate. ADH: I added precision to the text to read “... 3 and 5 replicas (with added replicas in more sensitive positions (101 mm, 85 mm and 54 mm) due to potential bottom and surface disturbance). L275-276 of the revised manuscript.
L255: name the vessel appropriately, i.e. Research Vessel Simon Stevin or RV Simon Stevin ADH: The change was made.
L291: Change sentence to “In the instance of a significant result, the one-way ANOVA was followed by the Tuckey HSD test to allow for subsequent pairwise comparison tests. ADH: The change was made.
Discussion
L369: change “set-up” to “design” ADH: The changes were made here and in other parts of the text.
L425: should be written in past tense “the aim was” and rephrased to better emphasise the point: “Finally the aim was to test the retention efficiency of substrates with living specimens, as a necessary step before recommending one particular substrate for in situ deployment” ADH: The change was made.
L426-428: As with the preceding sentence, I suggest “Therefore, we opted to perform these tests with L. conchilega as a means to validate the hypothesis emerging from results obtained in the previous ranking tests”. At this point, it may be necessary to confirm exactly what this hypothesis is you are mentioning. ADH: I changed the sentence as suggested and added a short summary of the hypothesis “... in previous ranking tests: thicker substrate with loose mesh size and 3D structure are good candidates to enhance larval settlement.”
L438: The mention of the presence of Phaeocystis needs context. If you are referring to DMS production, then please state so explicitly with a relevant reference. I suggest as a starting point: Liss, P.S., Malin, G., Turner, S.M., Holligan, P.M., 1994. Dimethyl sulphide and Phaeocystis: A review. Journal of Marine Systems 5, 41–53. https://doi.org/10.1016/0924-7963(94)90015-9 ADH: The presence of DMS due to Phaeocystis was a hypothesis, I modified the text to stay more general: “...with the dense presence of microalgae in the samples. Important algae blooms are frequent during the month of April in the North Sea [53], high chlorophyll-a concentrations were measured close to the Belgian coastline on both sampling days (European Organi-sation for the Exploitation of Meteorological Satellites, OLCI Level 2 CHL Concentration Daily Accumulated – Sentinel-3, on the 11/04/21 and 30/04/21).” L 518-522 of the reviewed manuscript.
L519-522: Is this not contradicting earlier statements? In L499-507 a case is made for how efficiently structure, like the application of sticks, are reducing flow, which in turn enhances attachment and settlement rate, while in L519-522 the authors link the superior performs of substrate G with the combined effects of increase flow and geotextiles. The premise in the latter hypothesis suggest higher flow should increase the number of larvae moving through the structure, which then equates to higher settlement and increased success of the substrate. Even so, it still contradicts the earlier statement. Which is best, low or higher flow? Surely the structure induced by the addition of sticks and the like would in fact reduce the flow of currents, thereby enhancing the ability of larvae to settle (further enhanced by the presence of vertical structures, mimicking their natural settling behaviour in the presence of tubes).
ADH: From our results, it seems that the geotextiles and the wooden sticks have a different effect on overlaying velocity. The geotextiles had a tendency to increase the velocity (L.584-585 of the revised manuscript), while the wood sticks had a tendency to decrease the velocity (L580-584 of the revised manuscript). To answer your question on which is the best flow, we hypothesized that the best flow is a reduced flow in order to increase the attachment rate but that still is dynamic to increase the encounter rate. I added this sentence in the conclusion to make it more clear “This optimal artificial substrate should be able to create a dynamic flow increasing the encounter rate between the larvae and the substrate while also reducing locally the velocity in order to favour the attachment rate of the larvae on the substrate. “ L639-642 of the revised manuscript.
One of the highest average overlaying velocities was measured with substrate G. This substrate performed the best in capturing both plastic particles and L.conchilega larvae. This result showed that a substrate combining both dynamic flow regimes and locally reduced velocity is the most efficient to enhance settlement. This result was quite surprising to us, as we initially only considered the reduction of flow velocity as a promotor of settlement. We concluded from our work that substrates combining both geotextile and wood sticks, like substrate G, must create a turbulent hydrodynamic flow creating the best conditions for settlement. Unfortunately, we did not have time to study properly turbulence and thus did not go into too much depth with this hypothesis.
